1. Field of the Invention
The present invention relates to improvements in methods and apparatus for sensing and characterizing small particles, such as blood cells or ceramic powders, suspended in a liquid medium having an electrical impedance per unit volume which differs from that of the particles. More particularly, it relates to improvements in methods and apparatus for sensing and characterizing such particles by the Coulter principle.
2. Discussion of the Prior Art
U.S. Pat. No. 2,656,508 to Wallace H. Coulter discloses a seminal method for sensing particles suspended in a liquid medium. An exemplary apparatus for implementing such method is schematically illustrated in FIG. 1. Such apparatus comprises a dual-compartment dielectric vessel 6 which defines first and second compartments 6A and 6B separated by a dielectric wall 7. Each of the compartments 6A and 6B is adapted to contain, and is filled with, a liquid medium M. The particles to be sensed and characterized are suspended at an appropriate concentration in liquid medium M and introduced into compartment 6A through a suitable inlet port 8 formed therein. Wall 7 is provided with a relatively large opening 7A which is sealed by a thin wafer W made of a homogeneous dielectric material. A small through-hole formed in wafer W provides a conduit 10, which constitutes the only operative connection between compartments 6A and 6B. An appropriate vacuum applied to an outlet port 11 suitably formed in compartment 6B causes the particle suspension to flow from compartment 6A to compartment 6B through conduit 10, discussed in detail below. Each particle in the suspension displaces its own volume of the particle-suspending liquid M, and conduit 10 provides a consistent reference volume against which that displaced volume may be compared. If the dimensions of conduit 10 and the concentration of particles in the suspension are appropriately selected, particles can be made to transit the conduit more or less individually. Conduit 10 thus functions as a miniature volumeter, capable under suitable conditions of making sensible the liquid displaced by individual microscopic particles.
To enable convenient sensing of the liquid displacement occasioned by particles transiting the conduit, the particle-suspending liquid M is made to have an electrical impedance per unit volume which differs from that of the particles. The contrast in electrical impedance between particle and suspending liquid thus converts the volume of displaced liquid into a proportional change in the electrical impedance of the liquid column filling conduit 10. Excitation electrodes 15 and 16 are respectively positioned in compartments 6A and 6B and electrically connected to a source 17 of electrical current, whereby a nominal electrical current is caused to flow through conduit 10 simultaneously with the particle suspension. Consequently, passage of a particle through conduit 10 produces a pulsation in the current flowing through the conduit which is proportional to the volume of liquid displaced by the particle. An AC-coupled sensing circuit 19, also electrically connected to excitation electrodes 15 and 16, operates to sense the pulsations in current between these electrodes. Thus, as individual particles pass through conduit 10, sensing circuit 19 produces an electrical signal pulse having an amplitude which is characteristic of the particle volume. Additional circuits 20 further process the particle signal pulses to provide a count of particles exceeding some particular volumetric threshold or, via the elegant positive-displacement metering system disclosed in U.S. Pat. No. 2,869,078 to Wallace H. Coulter and Joseph R. Coulter, Jr., the particle concentration. The volumetric distribution of the particles may be conveniently characterized by causing current source 17 to provide a constant current and analyzing the particle pulses with multiple-thresholding sizing circuitry 21 as described in U.S. Pat. No. 3,259,842 to Wallace H. Coulter et al. Alternatively, if current source 17 is caused to provide combinations of excitation currents, including at least one source of high-frequency alternating current as discussed in U.S. Pat. Nos. 3,502,973 and 3,502,974 to Wallace H. Coulter and W. R. Hogg, an apparent volume reflecting the internal composition of certain particles may be similarly characterized. Such characterization results are displayed or recorded by appropriate devices 22. This method of sensing and characterizing particles, by suspending them in a liquid medium having an electrical impedance per unit volume which differs from that of the particles and passing the resulting particle suspension through a constricting conduit while monitoring the electrical current flow through the conduit, has become known as the Coulter principle.
Central to the Coulter principle is the volumeter conduit 10 which enables electrical sensing of particle characteristics by constricting both the electric and hydrodynamic fields established in vessel 6. Although conduits of general longitudinal section and either circular or rectangular cross-sections are considered in the '508 patent, in this patent's practical example the conduit is a pinpoint circular aperture formed in the wall of a closed glass tube disposed within a second vessel so that both particle suspension and excitation current flow in the direction of the aperture axis between the two vessels. Such small apertures formed directly in a containment vessel are difficult to manufacture to repeatable geometry and tolerance. A practicable alternative utilized separate wafers cut from capillary tubing and sealed over a somewhat larger opening so that the tubing conduit formed the only operative connection between the two vessels; however, the conduit geometry in such wafers was unstable if sealing were done by the glass-fusion methods required for reliable seals. Ruby or sapphire jewels developed as anti-friction bearings for precision mechanical devices retain their geometry during fusion to glass, have excellent dielectric and mechanical properties, are readily available in a range of geometries and sizes, and so were indicated for use as conduit wafers in U.S. Pat. Nos. 2,985,830 and 3,122,431 to Wallace H. Coulter et al. The aperture tube described in these patents has been widely adapted, e.g., vessel 6 in FIG. 1, and ruby or sapphire ring jewels are frequently used as the conduit wafer W constricting the opening (e.g., 7A in wall 7) between containment compartments. As shown in the enlarged view of conduit wafer W in FIG. 2, a traditional Coulter volumeter conduit 10 thus comprises a continuous surface or wall 30 of length L which defines a right cylindrical opening of circular cross-section and diameter D through a homogeneous dielectric material of thickness L. Due to material homogeneity, the electrical resistivity of conduit wall 30 surrounding the flows of suspension and current through the conduit is substantially axisymmetric and uniform in any longitudinal conduit section. Because of its historical development, conduit wafer W is often called an "aperture wafer", and the traditional Coulter conduit 10 in conduit wafer W is commonly referred to as a "Coulter aperture".
The '508 patent describes two important functional properties as depending on the dimensions of Coulter volumeter conduits such as 10 in FIG. 1, viz., the volumetric sensitivity and the masking of one particle by another during simultaneous passage through the conduit volume. In principle, maximum volumetric sensitivity is obtained when the dimensions of volumeter conduit 10 approximate the diameter of the largest particle in the suspension of interest. Practically, conduit diameter D must approach twice the maximum particle diameter to minimize risk of clogging, and conduit length L is usually made as short as possible to minimize coincidence artifacts due to two or more particles simultaneously transiting the conduit. For a given conduit geometry, coincidence effects are only dependent on particle concentration and can be limited by increasing sample dilution. Industrial applications require various conduit diameters D between 0.010 mm and 2.000 mm, but many medical and scientific applications can be satisfied with conduit diameters D between 0.030 mm and 0.200 mm. Conduits with length-to-diameter ratio L/D=1.2 have been found to provide a combination of characteristics useful in a variety of applications, but medical applications benefit from the faster sample-throughput rates obtainable with less-diluted samples. As noted in the aforementioned U.S. Pat. No. 2,985,830, conduits with L/D=0.75 have proven a practicable compromise; such conduits permit acceptable processing rates and volumetric sizing of particles having diameters from about two percent, up to about 80 percent, of the conduit diameter D. In many applications decreased coincidence volumes or improved volumetric sensitivity would be advantageous, but field properties in the vicinity of the volumeter conduit frustrate use of shorter conduit lengths.
The '508 patent does not anticipate the complicating field properties of volumeter conduits such as 10 in FIG. 1. Since issuance of the '508 patent in 1953, the Coulter principle has been applied to a variety of particle-characterization problems important in numerous medical, scientific and industrial disciplines, and much experience has been gained with Coulter volumeter conduits. Many studies have been published regarding their functional properties, e.g., Volker Kachel, "Electrical Resistance Pulse Sizing: Coulter Sizing", to which reference is recommended for additional information (FLOW CYTOMETRY AND SORTING, 2nd. ed., M. R. Melamed, T. Lindmo, and M. L. Mendelsohn, eds., Wiley-Liss, New York, 1990, pages 45 to 80). Characteristics of signal pulses generated by particles passing through such conduits result from a complex interaction of the particles with both the electric field established in the liquid medium M by the current between excitation electrodes 15 and 16 and the hydrodynamic field established by the suspending liquid M carrying the particles through the conduit. Potential distributions for both fields show axisymmetric, semi-elliptical equipotentials at the entry orifice of the conduit, and for both fields, concentric flow converges toward this orifice. While the current through conduit 10 produces an electric field which is symmetric about the conduit midpoint as shown in FIG. 2, kinematic viscosity of the particle-suspending liquid causes a more complicated suspension flow through conduit 10 into compartment 6B. Some field properties of volumeter conduits relevant to the present invention may be summarized as follows:
1. As shown in the longitudinal section of conduit wafer W in FIG. 2, the particle-sensitive zone Z functionally includes not only the geometric volumeter conduit 10 defined by wall 30 but also the two semielliptical ambit electric fields 31 and 32 coaxial with, and outside the opposing ends of, the geometric conduit; the scale of these ambit fields depends only on the diameter D of the respective entrance and exit orifices, 33 and 34. In addition to producing current pulsations as they transit the geometric conduit, particles may also produce current pulsations if they pass through that portion of the suspending liquid M containing the ambit fields. PA0 2. The electric field forming that portion of particle-sensitive zone Z within the geometric volumeter conduit 10 is inhomogeneous, only approaching homogeneity at the conduit midpoint for L/D ratios of 2.0 or greater. Midpoint field inhomogeneity introduces errors of two types into particle pulses, viz., particles transiting the conduit along axial trajectories fail to generate fully-developed pulse amplitudes for conduits with L/D ratios less than 2.5, and particles with similar contrasts generate pulse amplitudes depending on the radial position of the particle trajectory, regardless of the L/D ratio of the conduit. Further, as will be discussed in Item 4, particles transiting the annular region containing the intense gradients at orifices 33 and 34 generate pulses having anomalous characteristics. This region, from conduit wall 30 inward to a radius r=0.75(D/2) for typical Coulter volumeter conduits, also defines the maximum particle diameter for which linear volumetric response is obtained. PA0 3. Conduit hydrodynamics determine particle presentation to particle-sensitive zone Z and, therefore, characteristics of the pulse generated by a given particle as it transits the geometric volumeter conduit. In response to the driving pressure gradient, the particle suspension in the sample compartment (6A in FIG. 1) develops concentric laminar flow accelerating toward volumeter conduit 10. At the entry orifice 33 in FIG. 2, the velocity profile of the constricting flow is quasi-uniform and of a magnitude determined by the desired sample volume, the time allowed to process it, and the cross-sectional area of the conduit. The flow just inside the conduit includes a shear layer at conduit wall 30, and particularly for L/D ratios less than about 3.0, the flow profile depends on the edge sharpness of entry orifice 33 and on how closely the kinematic viscosity of the suspending liquid permits it to follow the orifice geometry. When curvature of the edge at orifice 33 is sufficiently gradual, viscosity causes a transition from a quasi-uniform velocity profile toward the parabolic velocity profile of laminar flow (for practical reasons, orifice edges are usually sharp, and the shear layer surrounding the developing laminar flow may thicken to appreciably constrict the apparent flow cross-section). As is known in the fluidic art, for a circular conduit having a given L/D ratio, the degree of laminarity .xi. in a developing profile is inversely proportional to the Reynold's number e, i.e., .xi..varies.x/(R e), where x is the distance into the conduit from the entry orifice and R=D/2. Standard fluidic methods permit calculation of the differential volumetric flowrates through given annular cross-sections of conduit 10 centered on any particular radius. The results of such calculations for a typical suspending liquid M are shown in FIG. 3 for conduits with L/D=0 (a), 0.75 (b), 1.20 (c), 3.60 (d) and .infin. (e); here, (e) illustrates fully developed laminar flow in an infinitely long conduit. Although conduits with L/D ratios of 3.6 provide significant laminarity (d), flow through the conduit approaches (e) only for L/D ratios significantly greater than 10. The most-frequent, or modal, particle trajectories occur at the radius r corresponding to the maximum value (dotted) of these differential volumetric flow characteristics. At entry orifice 33, L/D=0 as in (a), and the modal particle trajectory thus occurs at r=(D-p)/2, or typically within a particle diameter p of conduit wall 30. For small particles, the entrance modal trajectory thus coincides with the outer shear layer of the quasi-uniform flow profile. Regardless of the sharpness of the edge of the entry orifice 33, flow at the exit orifice 34 is jetting flow (into the receiving compartment 6B in FIG. 1), with a toroidal low-pressure region surrounding the jet and overlapping the exit ambit field 32. As shown in FIG. 3, for particles exiting orifice 34 in FIG. 2, the modal trajectories occur in annuli centered at radii r=0.82(D/2) or 0.76(D/2) for conduits with L/D=0.75 (b) or 1.20 (c), respectively, and significant numbers of particles transit the conduit outside r=0.75(D/2), through the orifice gradients of the sensitive zone. The combination of a sharp edge at orifice 33 and the low L/D ratios of typical volumeter conduits also minimizes the stabilizing effect of viscosity, and as a consequence, both the through-flow and jetting patterns are sensitive to imperfections in the edge of entry orifice 33. Conduit L/D ratios of 2.0 or greater result in both smoother flow through the geometric volumeter conduit and less turbulence in the jetting zone outside the exit orifice; exit modal trajectories for such conduits are centered inside r=0.725(D/2). PA0 4. In volumetric applications of Coulter volumeter conduits, the most significant hydrodynamic effects are those on particle trajectory, shape, and orientation during passage through the particle-sensitive zone. As has been noted, the sensitive zone Z extends outward about one conduit diameter D from the entry orifice 33 in FIG. 2 and is overlapped by the convergent flow into conduit 10. Particles P in the sample vessel are entrained in the constricting flow and accelerated toward the entry orifice 33. As they enter the entry ambit 31 of the sensitive zone, particles on near-axial trajectories (e.g., trajectory A.sub.T) may be deformed by the pressure field, and nonspherical particles will be oriented with their long dimension parallel to flow; such particles generate pulses similar to the pulse of FIG. 4A. Particles entering the sensitive zone outside an axial cone approximately 50 degrees in half-angle will, in addition, be accelerated around the edge at orifice 33 and through the conduit in the annulus near wall 30 containing the intense orifice gradients. These orifice gradients cause particles on trajectories such as B.sub.T in FIG. 2 to generate M-shaped pulses (e.g., pulse of FIG. 4B) of anomalous amplitude (e.g., amplitude B) and duration due to gradients in, respectively, conduit field and liquid flow. Particles on an intermediate trajectory (e.g., C.sub.T in FIG. 2) may generate asymmetric pulses, such as the pulse in FIG. 4C, which demonstrate anomalous amplitude (e.g., amplitude C) only on their leading edge. The frequency of such pulses depends on the portion of the conduit cross section occupied by the orifice gradients and the average radial position of the modal trajectories, which in turn is determined by the length L of the conduit. Moreover, decelerating particles that have exited the geometric volumeter conduit 10 may be drawn back into the exit ambit 32 (e.g., trajectory D.sub.T in FIG. 2) as the suspending liquid recirculates into the toroidal low-pressure region surrounding the exit jet; if so, they generate extraneous pulses of low amplitude and long duration as shown by the pulse of FIG. 4D. Both recirculation and wall trajectories have adverse consequences significant in many applications of the Coulter principle, as illustrated in FIG. 5. In contrast to an ideal volumetric distribution 40, the recirculating particles (e.g., trajectory D.sub.T in FIG. 2) result in a secondary distribution 41 in the actual sample distribution 43; this spurious distribution reduces dynamic volumetric range and, for polydisperse samples, may altogether preclude analysis of the smaller particles. Due to their anomalous pulse amplitudes, particles following wall trajectories (e.g., B.sub.T and C.sub.T in FIG. 2) introduce artifactual high-volume skewness 42 into the actual sample distribution 43, so degrading system ability to resolve particles of nearly identical volumes. Conduits with L/D=3.3 have been shown to reduce skewness inaccuracies; then, exit modal trajectories are centered inside r=0.66(D/2). PA0 1. The ambit electric fields of the particle-sensitive zone resulting from the excitation current are substantially smaller, thereby reducing the likelihood of particle coincidence while increasing volumetric sensitivity; PA0 2. The cross section of the particle-sensitive zone containing substantially homogeneous field regions is significantly increased, thereby reducing the frequency of anomalous pulses and increasing the range in particle diameter for which the dynamic response is linear; PA0 3. The suspension flow profile through the particle-sensitive zone is quasi-laminar rather than quasi-uniform, whereby the proportion of particles per second transiting the substantially homogeneous areas of the particle-sensitive zone is increased, further reducing the frequency of anomalous pulses; and PA0 4. Particles are prevented from transiting the particle-sensitive zone on trajectories curving through the ambit electric fields, thereby eliminating both anomalous pulses due to particles entering the sensitive zone on high-angle trajectories and extraneous pulses due to exiting particles recirculating into the exit ambit field. PA0 A. Facilitating subsystems related to features 9, 12, 23, 24, and 25 in FIG. 1 may be eliminated, with significant reduction in manufacturing costs and appreciable improvement in system reliability but without important data inaccuracies; PA0 B. Because no auxiliary fluidic subsystems are required, particle concentration may be readily determined by positive-displacement volumetric methods; PA0 C. Because functional sample dilution due to sheath fluid is eliminated and need for post-collection pulse deletions can be significantly reduced, sample volumes and processing times may be reduced; and PA0 D. Because of the significantly reduced coincidence volume compared to the Coulter volumeter conduit, the rate of sample throughput can be increased for a given detectability threshold and level of coincidence artifact, or a larger conduit diameter may be used to decrease clogging concerns.
Consequently, the semielliptical equipotentials corresponding to the desired detectability threshold determine the effective spatial extent of the ambit fields 31 and 32. It can be shown that the portion of the particle-sensitive zone occupied by the geometric conduit 10 is (L/D)/(L/D+16K/3), where K is the product of the three diameter-normalized intercepts of the chosen threshold equipotential on a coordinate system with origins at the particular orifice center. It has been demonstrated that the effective ambit fields extend outward from the respective entrance and exit orifices 33 and 34 of volumeter conduit 10 approximately one conduit diameter D, with lateral intercepts at 1.15D, if pulse amplitudes from peripheral passages are to be limited to one percent of the theoretical maximum signal-pulse amplitude. For these one-percent equipotentials 35 and 36, the axial length of sensitive zone Z is (L+2D), K=1.3225, and for L/D=1.2 more than 85 percent of the particle-sensitive zone is external to the geometric Coulter conduit 10. The spatial extent of the sensitive zone increases the likelihood of particle coincidence, requiring greater sample dilution and processing times. In addition, the spatial extent of sensitive zone Z limits pulse signal-to-noise ratios, and therefore particle detectability, in two ways. First, particle contrasts and so pulse amplitudes are limited, since the volumetric sensitivity depends on the ratio of liquid volume displaced by each particle to the volume of liquid in the sensitive zone; and secondly, the noise tending to mask particle contrasts is increased, since it originates thermally throughout this latter volume. In principle, shorter conduit lengths L can decrease particle coincidence, increase conduit volumetric sensitivity, and decrease thermal noise; in practice, the benefits of decreasing conduit length are limited because, as L approaches zero, sensitive zone Z collapses to the ambit ellipsoid with volume determined by the conduit diameter and the desired threshold of pulse detectability.
Initially, apparatus based on the Coulter principle proved so extremely useful that data inaccuracies due to these functional conduit properties were tolerated. Gradually, however, data artifacts have become unacceptable impediments, particularly in applications where highly automated implementations are desirable, and so have prompted a broad variety of prior-art techniques intended to improve the accuracy of Coulter apparatus. This facilitating art will be summarized, for two purposes: Firstly, to illustrate the real difficulty in acceptably automating the Coulter principle, and secondly, to emphasize the advantages of the present invention. Such facilitating techniques include ones involving only the volumeter conduit, ones integrating the conduit into a subassembly, or those applying post-collection processing methods to the particle data. Some of this facilitating art has led to a requirement for one or more of the following in FIG. 1: a flow director 9, a second inlet port 12, and additional signal processing circuits 23, 24, and 25, each of which will be discussed in connection with the relevant art.
As noted in Item 1) above, the spatial extent of sensitive zone Z in FIG. 2 defines the coincidence, sensitivity, and noise characteristics of a given Coulter volumeter conduit. Because the diameter D of conduit 10 is usually determined by clogging concerns and its minimum length L as a compromise between artifacts due to coincidence and field inhomogeneities, variations in conduit geometry have been investigated as a means of improving functional properties. In the '508 patent, longitudinal conduit profiles other than right circular cylinders were disclosed as a means of varying the electric field along the geometric conduit and so establishing a desired particle pulse-shape. Ring jewels with various longitudinal bore profiles are available and so have seen application as conduit wafers, typically to facilitate mechanical goals. One early example used a straight ring jewel with a single spherical cup at the exit (U.S. Pat. No. 3,266,526); other examples use similar jewels but with the spherical cup at the conduit entry (U.S. Pat. Nos. 3,638,677; 3,783,376; 4,710,021; 5,150,037; 5,402,062; and 5,432,992). Such jewels retain sharp orifices due to the large radius of their spherical cups and so are functionally indistinguishable from the art first taught in U.S. Pat. No. 2,985,830. Functional improvement may be gained through a better fluidic match between the concentric entry flow and the quasi-uniform flow inside the entry orifice. Conduits which achieve this by mechanically limiting the off-axial extent of both the electric and fluidic fields at one or both orifices are described in U.S. Pat. No. 3,628,140 to W. R. Hogg and Wallace H. Coulter. Here, a jetting nozzle including a conical cup with half-angle of about 45 degrees is used to couple one or both conduit orifices to the adjacent volume of liquid. Although the patent attributes the resulting volumetric improvement to focusing of the excitation current, a more probable explanation lies in the observation, noted in Item 4) above, that particles entering the conduit within an axial cone of half-angle less than 50 degrees avoid the most intense artifactual effects of both conduit fields. The concept of the conical profile has also been adapted to conduits for use with optical sensing modalities, e.g., square or circular cross sections are described in U.S. Pat. No. 4,348,107 to R. C. Leif or U.S. Pat. No. 4,515,274 to J. D. Hollinger and R. I. Pedroso, respectively. Such conduits of square and triangular cross section, and techniques for constructing them by assembling multiple truncated dielectric pyramids, have been described (U.S. Pat. Nos. 4,673,288 and 4,818,103). Mechanical restriction of the conduit fields also decreases the volume occupied by the conduit ambits, with attendant improvement in the coincidence, noise, and recirculation characteristics of the conduit; an extreme form of this approach (U.S. Pat. No. 4,484,134 to M. T. Halloran) is discussed below. In U.S. Pat. No. 5,623,200, longitudinal profiles are described as a method of reducing magnitudes of the orifice gradients. Typically, however, pulse rise-times suffer due to the gradual change in cross section, and acceptable pulse characteristics usually require that the tapered section(s) be blended into a spherical cup centered on the conduit orifice, as is also taught in U.S. Pat. No. 3,628,140. In U.S. Pat. No. 3,733,548 to Wallace H. Coulter and W. R. Hogg, a semicircular longitudinal profile is described as producing better electric-field uniformity than the original Coulter conduit and, in principle, should also offer significant inlet flow matching. Yet another design (U.S. Pat. No. 3,739,258) primarily addresses flow matching, through use of a trumpet-shaped inlet to reduce thickening of the entry shear layer. Neither of the latter conduits significantly improves limitations due to the conduit ambit fields, and without further augmentation none of the above-discussed profiles yield data sufficiently artifact-free to be of wide use. In typical wafer dielectrics, all such shaped conduits are difficult to manufacture to practicable precision, and so all are expensive to produce. In some applications they may worsen the clogging problem.
Particle coincidence degrades count data directly through lost particle pulses. It also degrades volumetric data indirectly through inappropriate inclusion of misshapen pulses in the volumetric distribution. In some applications, adaptive dilution may acceptably limit coincidence artifact (U.S. Pat. No. 3,979,669 to T. J. Godin), or adaptive extension of the counting period may acceptably compensate it (U.S. Pat. No. 4,009,443 to Wallace H. Coulter et al.); but the resulting variable processing times are undesirable in many applications. In principle, the pulse loss due to coincidence can be predicted statistically, and many post-collection corrective techniques, e.g., coincidence-correction circuit 23 in FIG. 1, have been described in the scientific and patent literature; see, e.g., U.S. Pat. No. 3,949,197 to H. Bader for a review and example.
Other approaches estimate pulse loss based on pulse occurrence rate, count, or duration, e.g., U.S. Pat. No. 3,790,883 to P. Bergegere; U.S. Pat. No. 3,936,739 and U.S. Pat. No. 3,940,691 to W. R. Hogg; U.S. Pat. No. 3,949,198 to Wallace H. Coulter and W. R. Hogg; and U.S. Pat. No. 3,987,391 to W. R. Hogg. Limitations of several are discussed in U.S. Pat. No. 4,510,438 to R. Auer, which proposes correction for the actual coincidence rate as determined by an independent optical sensing modality. These methods may acceptably correct count data for coincidence pulse loss when automated for specific applications, but only those which inhibit incorporation of misshapen pulses can improve the population volumetric distribution. All add to design complexity, and some require extensive computational resources.
The volumetric sensitivity and noise characteristics of Coulter volumeter conduits limit dynamic measurement range, particularly for smaller particles. Noise originates by two mechanisms, heating noise resulting from dissipation of the excitation current in the resistance of the particle-sensitive zone, and Johnson noise generated in this resistance. These limit the maximum practicable excitation current, on the one hand, and fundamental particle detectability on the other. In the prior art, heating noise has been reduced by providing thermally conductive paths leading away from conduit 10. U.S. Pat. No. 3,361,965 to Wallace H. Coulter and Joseph R. Coulter, Jr., describes one such structure, in which one electrode is formed as a plated metallic coating on the outer surface of the aperture tube. In U.S. Pat. No. 3,714,565 to Wallace H. Coulter and W. R. Hogg the electrical path length through the suspending liquid, and so the thermal noise, is reduced by replacing the second electrode with a metallic element either composing, or coated onto the inner surface of, the aperture tube wall. The thermal effects are described more fully in U.S. Pat. No. 3,771,058 to W. R. Hogg; here, volumeter conduit 10 is formed in a wafer of thermally conductive dielectric and thermally connected to remote cooler regions via electrically and thermally conductive metallic coatings extending onto both planar surfaces of the conduit wafer. In U.S. Pat. No. 4,760,328 the same geometry is described in a structure which integrates sensing electronics onto the sapphire wafer. In all four of these patents the conductors cover extensive areas of the structure and variously approach volumeter conduit 10, but do not extend so close to the conduit as to interact with the effective ambit fields of its particle-sensitive zone Z. However, in U.S. Pat. No. 3,924,180 the Coulter conduit structure is modified by incorporation of thin conductors into the conduit structure, contiguous to conduit orifices 33 and 34, so forming potential-sensing electrodes in a dielectric sandwich through which the conduit penetrates; the intent is to minimize noise contributions to the sensed particle signal from the liquid outside the conduit ambits 31 and 32. Other techniques attempt to minimize noise effects, as for example the noise discriminator described in U.S. Pat. No. 3,781,674 to W. A. Claps, or the averaging of signals from tandem conduit/electrode structures similar to those of U.S. Pat. No. 3,924,180 to reduce Johnson noise as described in U.S. Pat. No. 4,438,390 to W. R. Hogg. In critical applications, certain of these may reduce heating noise generated in the conduit, but none significantly improves the volumetric sensitivity. U.S. Pat. Nos. 3,924,180 and 4,438,390 are the subject of further discussion, to follow.
As noted in Item 4) above, the effective sensitivity of Coulter volumeter conduit 10 may be further limited by the effects of exiting conduit flow carrying particles back into exit ambit field 32 of sensitive zone Z, e.g., trajectory D.sub.T in FIG. 2. These decelerating particles pass through the intense orifice field gradients and in many polydisperse samples result in long pulses of amplitudes comparable to those produced by the smaller particles. Unless precautions are taken to reduce the effects of these recirculating particles, both conduit sensitivity and usable dynamic range are degraded. In addition, when pulse-height techniques are used to develop volumetric distributions the pulses from recirculating particles cause extraneous peaks and broadening of actual particle distributions. At cost of reduced sample throughput, recirculation pulses may be excluded by pulse gating, e.g., by recursor pulse-edit circuit 24 in FIG. 1, either through analysis of pulses from the standard conduit or in response to a thin auxiliary detection electrode located in the conduit's geometric cylinder (U.S. Pat. No. 4,161,690). Longitudinal conduit profiles can mechanically reduce the liquid volume available to such particles and may be beneficial in some applications, as noted in the aforementioned U.S. Pat. No. 3,628,140 to W. R. Hogg and Wallace H. Coulter. Other applications are more critical, and many subassemblies incorporating the volumeter conduit have been described which attempt to prevent particles from recirculating into the conduit ambit fields. These either structure the exit flow path so that particles are mechanically prevented from re-entering the sensitive zone (U.S. Pat. Nos. 3,299,354 and 3,746,976 to W. R. Hogg or U.S. Pat. No. 4,484,134 to M. T. Halloran), use auxiliary fluidic circuits to dynamically sweep exiting particles away from the exit orifice (U.S. Pat. No. 4,014,611 to R. O. Simpson and T. J. Godin) or combine these two approaches (U.S. Pat. No. 3,902,115 to W. R. Hogg et al. and U.S. Pat. No. 4,491,786 to T. J. Godin, which contains a review of such methods). Other implementations have also been described (U.S. Pat. Nos. 4,253,058; 4,290,011; 4,434,398; 4,710,021; 5,402,062; 5,432,992; and 5,623,200). The dynamic sweep-flow method is widely used and involves metering appropriate volumes of the liquid medium M through a second inlet port 12 in FIG. 1, whereby the particles exiting conduit 10 are swept out of exit ambit field 32. These complex subassemblies can essentially eliminate recirculating particles, may include shaped conduits, and often include additional structure addressing effects of particles following wall trajectories. However, the large fluid volumes required for effective sweep-flow make it impractical to volumetrically determine particle concentration by positive-displacement methods. The approach taken by M. T. Halloran (in the aforementioned U.S. Pat. No. 4,484,134) potentially avoids need for auxiliary fluidic circuits and structures, by extending the insulating discs of U.S. Pat. No. 3,924,180 into an elongate tubular configuration; of an inner diameter substantially equal to that of the volumeter conduit, such extensions mechanically prevent recirculation of exiting particles into the exit ambit of the conduit and when carefully constructed can provide fluidic advantages of long conduits. However, for many applications of the Coulter principle such structures require complex mechanical designs difficult to construct to the necessary precision and tend to clog in use, due to their fluidic length.
The effective resolving ability of Coulter volumeter conduit 10 is determined by the hydrodynamic effects discussed in Item 4) above, specifically those carrying particles through the geometric conduit near its wall 30. The resultant characteristic M-shaped pulses (e.g., those in FIG. 4B or 4C) produce artifacts in the volumetric distribution, the importance of which is attested by the large amount of remedial prior art addressing them. This art is divided between two approaches, the early post-collection one of excluding the M-shaped pulses from the processed data and the later direct one of hydrodynamically controlling presentation of particles to the sensitive zone of the conduit. The electric field in the conduit sensitive zone Z, and particle trajectories (e.g., BT or CT) through it which produce problematic pulses, are illustrated in FIG. 2. Deletion of such pulses from the volumetric distribution data is suggested in U.S. Pat. No. 3,668,531 to W. R. Hogg, from which FIG. 2 is adapted. Other approaches have been described (U.S. Pat. Nos. 3,700,867 and 3,701,029 to W. R. Hogg; U.S. Pat. Nos. 3,710,263 and 3,710,264 to E. N. Doty and W. R. Hogg; U.S. Pat. No. 3,783,391 to W. R. Hogg and Wallace H. Coulter; U.S. Pat. No. 3,863,160 to E. N. Doty; and U.S. Pat. No. 3,961,249 to Wallace H. Coulter), all of which incorporate gating circuitry (25 in FIG. 1) responsive to various anomalous parameters of the misshapen pulses by which these pulses may be deleted from the pulse train processed for population distributions. Some of these are discussed in U.S. Pat. No. 3,863,159 to Wallace H. Coulter and E. N. Doty and in U.S. Pat. No. 4,797,624 to H. J. Dunstan et al., either of which well illustrates such gating methods. Gating may also be done in response to a detection signal from an auxiliary electrode (U.S. Pat. No. 4,161,690). The complexity of working implementations encouraged other approaches, and a simple flow-aligning device in front of the Coulter conduit was shown to improve volumetric accuracy (U.S. Pat. Nos. 3,739,268; 4,290,011; and 4,434,398). Further improvement was gained by injecting the particle stream directly into the conduit through an auxiliary flow director (U.S. Pat. No. 3,793,587 to R. Thom and J. Schulz and U.S. Pat. No. 3,810,010 to R. Thom), a technique now known as hydrodynamically focused flow. If in FIG. 1 the particle suspension is introduced through flow director 9 while liquid medium M is appropriately metered through port 8, the particles entering compartment 6A will be entrained into a sheath of the liquid medium M and carried through conduit 10 in the core of the composite flow pattern, with two important consequences. Firstly, the directed flow pattern prevents particles entering conduit 10 on trajectories such as B.sub.T and C.sub.T in FIG. 2, thereby eliminating pulses such as those in FIGS. 4B and 4C. Secondly, all particles transit conduit 10 inside the sheath liquid, which serves to center the particle trajectories inside the cross section of conduit 10 having relatively homogeneous electric fields, further reducing occurrence of anomalous particle pulses such as the pulse of FIG. 4B. Numerous conduit subassemblies incorporating focused flow have been described (e.g., U.S. Pat. No. 4,014,611 to R. O. Simpson and T. J. Godin; U.S. Pat. No. 4,395,676 to J. D. Hollinger and W. R. Hogg; U.S. Pat. No. 4,484,134 to M. T. Halloran; U.S. Pat. No. 4,515,274 to J. D. Hollinger and R. I. Pedroso; and U.S. Pat. No. 4,525,666 to M. R. Groves; U.S. Pat. Nos. 3,871,770; 4,165,484; 4,253,058; 4,760,328; 5,150,037; and 5,623,200), some of which also include provisions addressing recirculating particles and the best of which can yield nearly ideal volumetric distributions. All add complexity to practical apparatus, and the large fluid volumes required for effective sample focusing make it impractical to volumetrically determine particle concentration by positive-displacement methods. Because the entraining sheath flow restricts the sample stream to a small central portion of the geometric volumeter conduit, a functional concentration of particles within this volume occurs and limitation of coincidence effects typically requires use of lower particle concentrations than with unfocused systems.
Nearly all of the prior art concerns traditional volumeter conduits and the two-terminal implementation of the Coulter principle described in the '508 patent, but this simple form has been elaborated in, e.g., U.S. Pat. Nos. 3,924,180; 4,438,390 to W. R. Hogg; and U.S. Pat. No. 4,484,134 to M. T. Halloran. The fluidic advantages of long conduits (i.e., ones having L/D.gtoreq.2) have long been known, but the large coincidence volumes and noise levels associated with such conduits limit their practical usefulness. As has been discussed, in U.S. Pat. No. 3,924,180 thin insulated electrodes are located along the volumeter conduit to enable four-terminal potential sensing of particle pulsations from a small portion of the actual conduit length as one way of minimizing these limitations, and in U.S. Pat. No. 4,438,390 the structure of U.S. Pat. No. 3,924,180 is replicated in a single structure to produce a plurality of potential-sensitive zones in tandem, the potentials sensed thereby being averaged as a means of reducing Johnson noise. In U.S. Pat. No. 4,484,134 the insulative structures covering the electrodes in U.S. Pat. No. 3,924,180 are extended into tubular form, as has also been discussed. The plural electrodes incorporated into the conduit structures of these three patents are required to be as thin as practicable, to avoid significant influence on the conventional electric fields resulting from the excitation current, and are electrically connected to external sensing apparatus. The liquid column in these structures thus forms a resistance divider across which the total particle pulse amplitude is developed, but across only that section separating the potential-sensing electrodes of which is the sensed particle signal developed. Consequently, the loss of signal pulse amplitude due to the voltage-dividing action of the liquid column may offset any decrease in noise. Although these patents allude to fluidic advantages of the longer conduit structures, neither they nor the other known prior art either detail the origin of such advantages or suggest a specific method whereby such advantages may be systematically obtained.
There is no question that the accuracy, resolution, and convenience of Coulter apparatus have substantially benefited through the teachings of the many patents cited above, and fully automated apparatus is now available in which functional properties of the Coulter volumeter conduit are acceptably compensated. The Coulter principle has gained worldwide acceptance, and many national standards include methods based on it. Apparatus incorporating the Coulter principle is now available from a number of manufacturers, and its economic importance is attested by the voluminous prior art which has developed around it. However, much of this art concerns methods which increase apparatus complexity, with attendant decreased reliability and increased costs throughout the design, production, and maintenance cycle. Most requires multiple precision components difficult of manufacture and assembly to the requisite accuracy. Ones requiring auxiliary fluidic subsystems preclude positive-displacement volumetric determinations of particle concentration. Ones based on post-collection data processing discard particle data, and so require greater sample volumes or longer sample processing times. Each only mitigates an undesirable consequence of particle/field interaction in the particle-sensitive zone of the Coulter volumeter conduit, rather than amending the underlying characteristics of the electric and hydrodynamic fields. Because the prior art addresses their consequences, rather than their origins, functional properties of Coulter volumeter conduits have evolved little since the issuance of the '508 patent in 1953.
Reliability and cost competitiveness have become increasingly important considerations in apparatus design, and it would be advantageous to achieve the performance now attainable through the prior art summarized above, but without the increased complexity and costs associated with this art. It would be preferable to obviate such facilitating methods and apparatus, by directly amending field characteristics of the volumeter conduit. It would be desirable that any solution to this long-standing need be directly substitutable for the Coulter volumeter conduit of existing methods and apparatus, e.g., those of U.S. Pat. Nos. 2,656,508 or 3,259,842. It would also be desirable that this solution permit volumetric determination of particle concentration by positive-displacement methods such as, e.g., those described in U.S. Pat. No. 2,869,078.